Báo cáo khoa học: Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna ppt

Molecular mass of macromolecules and subunits and the
quaternary structure of hemoglobin from the
microcrustacean Daphnia magna
Tobias Lamkemeyer
1,2
, Bettina Zeis
1
, Heinz Decker
3
, Elmar Jaenicke
3
, Dieter Waschbu
¨
sch
3
,
Wolfgang Gebauer
4
,Ju
¨
rgen Markl
4
, Ulrich Meissner
4
, Morgane Rousselot
5
, Franck Zal
5
,
Graeme J. Nicholson
6

and Ru
¨
diger J. Paul
1
1 Institut fu
¨
r Zoophysiologie, Westfa
¨
lische Wilhelms-Universita
¨
t, Mu
¨
nster, Germany
2 Proteom Centrum Tu
¨
bingen, Eberhard-Karls-Universita
¨
t, Tu
¨
bingen, Germany
3 Institut fu
¨
r Molekulare Biophysik, Johannes Gutenberg Universita
¨
t, Mainz, Germany
4 Institut fu
¨
r Zoologie, Johannes Gutenberg Universita
¨
t, Mainz, Germany

5 Equipe Ecophysiologie: Adaptation et Evolution Mole
´
culaires, UPMC–CNRS UMR 7144, Station Biologique, BP 74, Roscoff, France
6 Institut fu
¨
r Organische Chemie, Eberhard-Karls-Universita
¨
t,Tu
¨
bingen, Germany
Keywords
glycosylation; hemoglobin; macromolecule;
molecular mass; quaternary structure
Correspondence
T. Lamkemeyer, Interfakulta
¨
res Institut fu
¨
r
Zellbiologie, Proteom Centrum Tu
¨
bingen,
Eberhard-Karls-Universita
¨
t, Auf der
Morgenstelle 15, D-72076 Tu
¨
bingen,
Germany
Fax: +7071 295359

Tel: +7071 2970556
E-mail: Tobias.Lamkemeyer@uni-
tuebingen.de
(Received 23 March 2006, revised 17 May
2006, accepted 30 May 2006)
doi:10.1111/j.1742-4658.2006.05346.x
The molecular masses of macromolecules and subunits of the extracellular
hemoglobin from the fresh-water crustacean Daphnia magna were deter-
mined by analytical ultracentrifugation, multiangle laser light scattering
and electrospray ionization mass spectrometry. The hemoglobins from
hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-
poor Daphnia magna were analyzed separately. The sedimentation coeffi-
cient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass
was 583 kDa (hemoglobin-rich animals) determined by AUC and
590.4 ± 11.1 kDa (hemoglobin-rich animals) and 597.5 ± 49 kDa (hemo-
globin-poor animals), respectively, determined by multiangle laser light
scattering. Measurements of the hemoglobin subunit mass of hemoglobin-
rich animals by electrospray ionization mass spectrometry revealed a signi-
ficant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme
groups. The hemoglobin subunits are modified by O-linked glycosylation in
the pre-A segments of domains 1. No evidence for phosphorylation of he-
moglobin subunits was found. The subunit migration behavior during
SDS ⁄ PAGE was shown to be influenced by the buffer system used (Tris
versus phosphate). The subunit mass heterogeneity found using Tris buffer-
ing can be explained by glycosylation of hemoglobin subunits. Based on
molecular mass information, Daphnia magna hemoglobin is demonstrated
to consist of 16 subunits. The quaternary structure of the Daphnia magna
hemoglobin macromolecule was assessed by three-dimensional reconstruc-
tions via single-particle analysis based on negatively stained electron micro-
scopic specimens. It turned out to be much more complex than hitherto

proposed: it displays D4 symmetry with a diameter of approximately
12 nm and a height of about 8 nm.
Abbreviations
AUC, analytical ultracentrifugation; BN-PAGE, blue native polyacrylamide gel electrophoresis; ESI-MS, electrospray ionization mass
spectrometry; Hb, hemoglobin; MALLS, multiangle laser light scattering; MRA, multireference alignment; MSA, multivariate statistical
analysis; RuBPs, ruthenium II tris(bathophenanthroline disulfonate).
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3393
Invertebrate hemoglobins show very high structural
diversity in comparison with the uniform tetrameric
structure in vertebrates. They range from the 17 kDa
single-chain globins found in bacteria, algae, protozoa
and plants to the large multisubunit, multidomain
hemoglobins found in nematodes, molluscs and crusta-
ceans up to the giant annelid and vestimentiferan
hemoglobins of about 3600 kDa, which are composed
of globin and nonglobin subunits [1].
Specifically, the structure and function of the hemo-
globin (Hb) of the microcrustacean Daphnia magna
have been addressed by many studies over the last dec-
ades. Daphnia magna Hb is freely dissolved in the
extracellular fluid. It is a multisubunit Hb composed
of didomain globin chains. The synthesis of this Hb is
regulated by ambient oxygen concentration, tempera-
ture and juvenoid hormones [2–4]. Environmental
hypoxia, for example, may cause an increase of Hb
concentration in D. magna by a factor of 16, corres-
ponding to Hb concentrations between 55 and
888 lmol hemeÆL
)1
[5,6]. Concomitant changes of oxy-

gen affinity between 1.02 and 0.15 kPa (P
50
) have been
reported [7], which are related to Hb multiplicity (iso-
hemoglobins composed of different Hb subunits)
[1,8,9]. The biological advantages of increased Hb
concentration and oxygen affinity under hypoxia are
manifold [1,9]. Accordingly, Daphnia is currently the
focus of investigations aimed at the structure and evo-
lution of its globin genes as well as of its physiologic
adaptations [1].
Hb synthesis takes place in the fat cells and epipo-
dite epithelial cells of D. magna [10], which are the
only known sites of Hb synthesis in crustaceans [1].
The seven known Hb subunits (DmHb) [3,8,11] are
encoded by at least six Hb genes (dmhb) [12]. At least
four of them are organized in a cluster in the order
dmhb4, dmhb3, dmhb1 and dmhb2 [11]. On the basis of
nucleotide and derived amino acid sequences, the
molecular masses of Hb subunits are 36.228 kDa
(dmhb1), 36.177 kDa (dmhb2), 36.217 kDa (dmhb3)
(sequence data from [11]) and 35.921 kDa (dmhb4)
(sequence data from [12]). However, the molecular
masses of the Hb subunits experimentally determined
by gel electrophoresis were 36.2 (DmHbA–DmHbD),
37.9 (DmHbF, DmHbG) and 40.6 kDa (DmHbE) [8],
raising the question of the reason for this difference.
For the molecular mass of the native D. magna Hb
complex, reported values vary between 494 and
670 kDa, depending on the methods used (gel filtra-

tion, ultracentrifugation, gel electrophoresis [13–15]).
Suggesting very low molecular masses of about 31–
33 kDa for the Hb subunits and 494 kDa for the
native multimer, Ilan et al. [14] concluded that 16
polypeptide chains, each carrying two heme-binding
domains, form one D. magna Hb macromolecule.
From electron micrographs, two models of the three-
dimensional structure have been suggested [14]: (a) a
cyclic structure composed of all 16 subunits; and (b) a
dihedral structure, in which the subunits are grouped
in two layers stacked in an eclipsed orientation.
Accordingly, the present data on the molecular mass
of the native Hb complex as well as the Hb subunits
are inconsistent. In addition, there are only two hypo-
thetical models concerning the structure of the multi-
subunit assembly, which has stimulated further studies
on the structure of D. magna HB. Three-dimensional
reconstruction from transmission electron microscopy
promises to be a satisfactory way to describe the qua-
ternary structure of high molecular mass invertebrate
respiratory proteins [16–18].
To elucidate the important structural characteristics
of the extracellular Hb of D. magna, the molecular
mass of the native Hb complex as well as those of
denatured Hb subunits were determined by analytical
ultracentrifugation and multiangle laser-light scattering
(MALLS) or by MS and gel electrophoresis, respect-
ively, in normoxically (pale) and hypoxically (red)
raised D. magna. In addition, possible post-transla-
tional modifications were investigated. To investigate

the quaternary structure of D. magna Hb, transmission
electron microscopy and three-dimensional reconstruc-
tion of the macromolecule were carried out.
Results
The native Hb complex from the hemolymph of red
D. magna purified by chromatofocusing and diluted in
10 mm ammonium acetate buffer was subjected to
ultracentrifugation to determine the sedimentation
velocity (Fig. 1A). Use of the van Holde–Weischet
analysis on the data (Fig. 1B) showed that approxi-
mately 75% of the native protein sedimented with a
sedimentation coefficient of 17.4 ± 0.1 S (Fig. 1C).
The remaining proteins sedimented at 16.3–17.1 S.
Subsequent sedimentation equilibrium experiments
(Fig. 2) resulted in a molecular mass of 583 kDa for
the native Hb complex from the hemolymph of red
D. magna. Owing to their small quantity, the molecu-
lar mass of slower-sedimenting proteins could not be
analyzed with this method.
MALLS analyses of pale and red D. magna Hb
obtained by purification of crude animal extracts via
gel filtration or chromatofocusing provided molecular
mass determinations during the elution of peaks
(Fig. 3). On average, the molecular mass was
597.5 ± 49 kDa (n ¼ 3 samples: 562.3, 576.4 and
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3394 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
653.9 kDa; for each sample, the molecular mass is
averaged over 130 measured points) for pale D. magna
Hb and 590.4 ± 11.1 kDa (n ¼ 5 samples: 576.2,

582.2, 593.2, 596.0 and 604.2 kDa; for each sample,
the molecular mass is averaged over 130 measured
points) for red D. magna Hb. A second peak in case of
pale D. magna (Fig. 3A) was heterogeneously com-
posed (polydispersity), as indicated by the inclination
of individual measuring points. The molecular mass of
these proteins (Hb dissociation products or other pro-
teins) was 385 ± 9 kDa.
Using a multiphasic buffer system according to
Laemmli [19], gel electrophoresis (SDS ⁄ PAGE) resul-
ted in the separation of D. magna Hb subunits into
three different bands (Fig. 4A) with molecular masses
of 40.0, 38.1 and 35.1 kDa (pale D. magna) and 40.0,
36.9 and 35.1 kDa (red D. magna). As such separa-
tions may be due to a specific buffer system instead of
reflecting actual mass differences [20], the protocol of
Weber and Osborn [21] was employed: in this case,
only one band for both Hb from the hemolymph of
pale D. magna and Hb from red D. magna appeared
(Fig. 4B), corresponding to a molecular mass of
approximately 39.2 kDa. Bands with molecular masses
above 66 kDa may originate from undissociated Hb
molecules or impurities in the sample.
For an exact determination of the molecular mass of
D. magna Hb subunits, electrospray ionization mass
spectrometry (ESI-MS) analyses were performed. For
red D. magna Hb purified by gel filtration, an ion ser-
ies in the mass ⁄ charge ratio (m ⁄ z) range of 1500–3000
occurred under denaturing conditions (Fig. 5A). De-
convolution of the spectrum resulted in a single signifi-

cant peak which corresponded to a molecular mass of
36.482 ± 0.0015 kDa (Fig. 5B). The acidic conditions
used for ESI-MS analysis led to the dissociation of the
heme group (616.5 Da) from the polypeptide chains.
Thus, the final mass for the didomain subunit is
37.715 kDa, including two heme groups.
To test for post-translational modifications of red
D. magna Hb subunits, a staining technique specific
for glycosylated proteins was used (Fig. 6A), followed
by staining with ruthenium II tris(bathophenanthroline
disulfonate) (RuBPs) (Fig. 6B). All Hb subunit spots
and the glycosylated proteins of the CandyCane mar-
ker (Fig. 6C) were specifically labeled after staining for
glycoproteins. The remaining marker proteins became
visible only after silver staining for total protein. Obvi-
ously, D. magna Hb subunits are glycosylated.
These staining results are corroborated by enzymatic
deglycosylation of Hb subunits. After two-dimensional
gel electrophoresis, the Hb subunits A–D appear as a
train of spots, whereas the subunits E–G also differ in
molecular mass [3,8] (Fig. 7A). To test whether this
separation is influenced by subunit glycosylation, Hb
(purified by gel filtration) was incubated with different
sets of enzymes removing only N-linked sugars
(Fig. 7B), only O-linked carbohydrates (Fig. 7C) or
both types of glycans (Fig. 7D), respectively. The
untreated sample (Fig. 7A, control) represents the typ-
ical spot pattern of red D. magna Hb consisting of the
subunits A–D and F. The Hb pattern after incubation
radius (cm)

6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0
mn 08
2 ta
ecn
a
b
r
osba
0.0
0.1
0.2
0.3
0.4
0.5
0.6
A
time
-0.5
(min
-0.5
)
0.00 0.01 0.02 0.03 0.04
)S( tneiciffeoc noitatnemides
12
14
16
18
20
22
24

B
sedimentation coefficient (S)
16.0 16.5 17.0 17.5 18.0
noitartnecnoc latot fo %
0
20
40
60
80
100
C
time
center bottom
Fig. 1. Sedimentation velocity analysis of
the hemoglobin (Hb) of red (Hb-rich) Daph-
nia magna. A preparation of Hb purified by
chromatofocusing was centrifuged at
116 480 g at 20 °C; the protein concen-
tration in the cell was 1.0 mgÆmL
)1
in
10 m
M ammonium acetate buffer, pH 6.7.
(A) The absorbance at 280 nm along the
length of the cell was recorded every 4 min.
(B) The sedimentation coefficient was deter-
mined with the van Holde–Weischet extra-
polation plot. (C) The distribution plot shows
that approximately 75% of the protein sedi-
mented at 17.4 ± 0.1 S, whereas only a

small fraction sedimented more slowly
(16.3–17.1 S).
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3395
with N-glycosidase F (Fig. 7B) is obviously identical to
that of the untreated sample. Incubation with enzymes
specific for the removal of O-linked glycans led to the
occurrence of additional spots (marked with white cir-
cles in Fig. 7C) with a higher electrophoretic mobility.
Consequentially, these spots also emerged, when the
Hb sample was treated with the whole set of enzymes
removing N-linked and O-linked glycans (Fig. 7D).
Potential N-linked glycosylation sites (NXT ⁄ S) were
not found in the amino acid sequences of D. magna
Hb [22]. Prediction of O-linked glycosylation was per-
formed using the recently released NetOGlyc 3.1 server
[23], and the results indicated the pre A-segments of
domain 1 as sites for glycosylation events in all Hb
sequences. The numbers of possible glycosylation sites
exceeding the threshold (G-score ¼ 0.5) were 11 for
DHb 1 and DHb2 and 12 for DHb3 and DHb4,
respectively.
For identification of the carbohydrates bound to
D. magna Hb subunits, saccharides were released by
methanolysis and separated by GC followed by MS.
Chromatograms of the carbohydrates of D. magna Hb,
of the corresponding blank and of a mannose standard
are shown in Fig. 8. The dominant carbohydrate in
D. magna Hb is mannose, which was unambiguously
identified by the analysis of a mannose standard (gray-

shaded curve in Fig. 8). A lower, but still highly signi-
ficant, increase (compared to the blank; Fig. 8, inset)
in the intensity of galactose and glucose was also
observed (the dashed line in Fig. 8 represents the
)mn
08
2
(
ecnabrosba
0.0
0.2
0.4
0.6
0.8
1.0
III
II
I
center bottom
A
-0.05
0.00
0.05
slaudiser
-0.05
0.00
0.05
position, r
mp
2

- r
mc
2

(cm
2
)
0.0 0.5 1.0 1.5 2.0 2.5
-0.05
0.00
0.05
III
I
II
B
Fig. 2. Sedimentation equilibrium analysis of the hemoglobin (Hb)
of red Daphnia magna. A preparation of Hb purified by chromatofo-
cusing was centrifuged at 4660 (I), 5900 (II) and 7280 (III) g at
4 °C, until equilibrium was achieved (A); the protein concentration
was 1.0 mgÆmL
)1
in 10 mM ammonium acetate buffer, pH 6.7. The
results at all speeds were globally fitted, resulting in a molecular
mass of 583 kDa. The residuals (B: I–III) reflect the deviation of
individual measuring points from the calculated fit, indicating the
quality of the fit and the calculated molecular mass. mp, measuring
point; mc, meniscus.
Fig. 3. MALLS analysis of the hemoglobin (Hb) of (A) pale (Hb-poor)
and (B) red (Hb-rich) Daphnia magna purified by gel filtration or

chromatofocusing, during the elution on a gel filtration column (Su-
perose 6-C). The solid curve represents the refractive index signal
profile versus the elution volume, and the distribution of the
molecular weight values is represented by crosses. The latter infor-
mation yielded molecular masses of 597.5 ± 49 kDa (n ¼ 3 sam-
ples from different animal groups) for pale and 590.4 ± 11.1 kDa
(n ¼ 5 samples from different animal groups) for red D. magna Hb.
The samples from pale D. magna additionally showed a second
protein component of a lower molecular mass (384.5 kDa).
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3396 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
maximum total ion current of the blank: glucose,
about 2500).
The results of all experiments to demonstrate phos-
phorylation of Hb subunits, however, were negative.
Neither specific staining of phosphorylated proteins in
gels (Pro Q Diamond stain) nor detection by western
blotting using antibodies specific for phosphoserine
and phosphotyrosine residues or different MS methods
showed any indication of phosphorylation of D. magna
Hb subunits (data not shown).
To assess the organization of the native Hb com-
plex, electron microscopy and three-dimensional recon-
structions were employed. After staining with 2%
uranyl acetate, the highly concentrated Hb macromole-
cules in the hemolymph of red D. magna (Fig. 9C,D)
frequently showed specific clover-leaf structures
(marked by arrows). These characteristic clover-leaf
structures were scarcely found in the hemolymph of
pale D. magna (Fig. 9A,B). In addition to Hb mole-

cules, another type of protein was detected in the
hemolymph of pale and red D. magna (probably fer-
ritin, marked by asterisks in Fig. 9A,C,D).
Because of its higher hemolymph concentration and
higher degree of structural detail, red D. magna Hb
was considered to be more promising for single-parti-
cle image processing. As purification by gel filtration
did not completely remove the ferritin, which would
have interfered with image analysis, Hb was purified
by chromatofocusing, resulting in a small quantity of
ferritin in comparison to the large number of Hb mole-
cules in the samples. In addition, structures clearly
representing ferritin molecules were omitted from sub-
sequent image analysis. The macromolecules within a
sample were spread across the holes in a perforated
carbon support film, causing the molecules to have
many different axial orientations relative to the elec-
tron beam. As the three-dimensional organization of
subunits in a native Hb complex of D. magna is as yet
unknown, different symmetry features had to be tested
and verified. Therefore, the simplest symmetry with
minimum assumptions (C2; two-fold symmetry, bot-
tom and top of the molecule different) was chosen for
an initial estimation. From the established three-
dimensional model, it could be expected that the bot-
tom and the top of the molecule would be identical (D
symmetry). Manual and automated (anchor set) Euler
searches were then systematically performed for two-
fold to eight-fold D symmetries (D2–D8). For the Hb
of red D. magna, the class averages calculated from

electron micrographs seemed to suggest a tetrameric
(D4) symmetry (Fig. 9E). Moreover, the best corres-
pondence between class averages and reprojections was
found for this symmetry, so that D4 symmetry was
finally selected and used for three-dimensional recon-
structions. The preliminary three-dimensional model of
red D. magna Hb has a sub-30 A
˚
resolution (Fig. 9F).
In top view (Fig. 9F, center), the molecule has the
overall shape of a square. Each side of the square has
a length of approximately 12 nm. A single mass in the
center of the molecule was observed. However, this
apparent mass may be an artefact of the negative
staining procedure (see Discussion). In side views
(Fig. 9F, left and right), the molecule appears as a
compressed sphere with a height of about 8 nm. With-
out additional information such as X-ray data, the
molecule’s handedness cannot be defined, meaning that
it cannot be determined whether the real structure or
its mirror image was reconstructed.
Discussion
Molecular mass and sedimentation coefficient
of Hb macromolecules
Crustacean hemoglobins are large polymers with
molecular masses between 240 and 800 kDa, and sedi-
mentation coefficients between 11 and 19 S. Reported
B
14.2
66

45
36
29
24
20.1
phosphate buffer
Marker
(kDa) pale red
A
66
45
36
29
24
20.1
Tris buffer
Marker
(kDa) pale red
Fig. 4. The influence of the buffer system used for gel electrophor-
esis on the band pattern of pale and red Daphnia magna hemoglob-
in (Hb). The hemolymph of pale (2.0 lg of Hb) and red (2.0 lgof
Hb) D. magna was subjected to electrophoresis according to
Laemmli [19], using either Tris buffer (A) or sodium phosphate
buffer (according to Weber and Osborn [21]) (B). In the first case,
Hb was separated into three bands (pale D. magna, 40.0, 38.1 and
35.1 kDa; red D. magna, 40.0, 36.9 and 35.1 kDa), whereas in the
second case, only a single band at 39.2 kDa was detected.
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3397
values for the molecular mass of D. magna Hb vary

considerably (494–670 kDa).
Molecular mass determinations by gel filtration and
gel electrophoresis yielded values between 500 kDa
[24] and 670 kDa [15]. However, interactions between
protein molecules and matrix, whether beads (gel fil-
tration) or polyacrylamide ⁄ agarose (gel electrophor-
esis), may cause false estimations of molecular mass.
Actually, it is not the molecular mass, but the effect-
ive molecular radius, that determines the mobility of
1500 2000 2500 3000
m/z
tniytisne()%
2147.0
1921.1
1825.1
A
1659.3
2281.2
2433.2
2606.9
0
100
50
B
0
36000 36500 37000 37500 38000
molecular mass (Da)
36482
100
50

snetnIyti()%
Fig. 5. Mass spectra of red Daphnia magna
hemoglobin (Hb) purified by chromatofocus-
ing. (A) Charge state ESI-MS spectrum. (B)
MaxEnt-deconvoluted spectrum. Prior to
MS, the Hb samples had been desalted and
denatured by adding acetonitrile ⁄ water con-
taining 0.2% formic acid. In (A), seven of
the 10 peaks representing the subunit
monomer are labeled. In (B), the molecular
mass (Da) of the Hb subunit (without heme)
is given above the peak.
180
82
42
A
B
C
D
F
glycoprotein stain
A
B
RuBPs stain
A
B
C
D
F
C

97
66
29
18
Fig. 6. Staining for glycosylation of red
Daphnia magna hemoglobin (Hb). Hb puri-
fied by gel filtration (100 lg) was subjected
to two-dimensional gel electrophoresis and
a staining procedure specific for glycosylat-
ed proteins (A). Hb spots became visible
after staining for glycosylated proteins. Sub-
sequently, the gel was stained with ruthen-
ium II tris(bathophenanthroline disulfonate)
(RuBPs) for total protein analysis (B). To
prove the specificity of the staining tech-
nique, the CandyCane molecular mass mar-
ker (C) consisting of glycosylated and
nonglycosylated proteins in alternating order
was stained for glycoproteins (left lane, bold
italicized digits: after staining with Pro Q
Emerald 488) and total protein (right lane:
after silver staining).
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3398 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
a macromolecule during gel filtration [25]. The accu-
racy of SDS ⁄ PAGE is also limited to about 10–
40%, because of, for example, unusual amino acid
composition, glycosylation or phosphorylation
[21,26,27]. Covering the charge of native proteins by
Coomassie Brilliant Blue during gel electrophoresis

(blue native PAGE; BN-PAGE) resulted in a
molecular mass of about 600 kDa in the case of
native D. magna Hb (data not shown).
During analytical ultracentrifugation, protein
molecules are freely dissolved and no interactions
with a matrix take place. Provided that the partial
specific volume of a protein analyzed is exactly
known, the error of measurement is below 3% [20].
Using the partial specific volume of D. magna Hb
(0.749 mLÆg
)1
; measured by Ilan et al. [14]), sedimen-
tation equilibrium experiments on red D. magna Hb
(Figs 1 and 2) revealed a molecular mass of 583 kDa.
The value used for the partial specific volume is sim-
ilar to those of other invertebrate hemoglobins such
as those of Caenestheria inopinata (0.747 mLÆg
)1
[28]),
Lepidurus apus lubbocki (0.745 mLÆg
)1
[29]), Lumbri-
cus terrestris (0.740 mLÆg
)1
[30]), Planorbis corneus
(0.745 mLÆg
)1
[31]), and Triops longicaudatus
(0.743 mLÆg
)1

[32]). The measured sedimentation coef-
ficients were comparable (between 17.4 and 17.8 S) in
all studies on D. magna Hb [13,14] (this study). How-
ever, the molecular mass suggested by Sugano and
Hoshi [13] for D. magna Hb (670 kDa) was not deter-
mined by sedimentation equilibrium experiments, but
was deduced from the measured sedimentation coeffi-
cient (17.8 S). As they found identical sedimentation
coefficients for D. magna and Moina Hb, they conclu-
ded that the molecular masses of D. magna and
Moina Hb (molecular mass: 660–670 kDa, determined
by ultracentrifugation [33]) were identical. Previous
sedimentation equilibrium experiments on D. magna
Hb [14] gave a molecular mass of 505 ± 35 kDa in a
first experiment and one of 483 ± 27 kDa in a sec-
ond experiment, resulting in an overall molecular
mass of 494 ± 33 kDa. For the subunits of D. magna
Hb, the reported molecular mass (about 31 kDa; also
determined by ultracentrifugation) was distinctly
lower than that from the ESI-MS data of our study
(37.715 kDa) or the value deduced from amino acid
sequence (36.2 kDa). Accordingly, molecular masses
seemed to be generally underestimated in that previ-
ous ultracentrifugation study, which may be due to
the buffer system used (0.1 m sodium phosphate,
pH 6.8).
To additionally verify the results from the ultra-
centrifugation experiments, the molecular mass of the
D. magna Hb complex was determined by MALLS
(Fig. 3). Zhu et al. [34] have shown that the deter-

mination of molecular mass by MALLS could have
an error of measurement below 2%. The determined
values (590.4 ± 11.1 kDa for red D. magna and
597.5 ± 49 kDa for pale D. magna) agree well with
the data from ultracentrifugation (583 kDa for red
D. magna). The second peak in case of pale
D. magna Hb could be a dissociation product of Hb,
as partial dissociation of D. magna Hb at pH 9–10
yielded a 353 kDa fragment determined by gel filtra-
tion [35].
control
A
1
A
2
B
1
B
2
C
1
C
2
D
1
D
2
F
1
F

2
*
A
de-N
A
1
A
2
B
1
B
2
C
1
C
2
D
1
D
2
F
1
F
2
*
B
de-O
*
C
de-N/O

*
D
Fig. 7. Mobility shift assays after enzymatic deglycosylation of
Daphnia magna hemoglobin (Hb). To determine the type of glycosy-
lation (N- or O-linked), Hb was deglycosylated using different sets
of enzymes, and this was followed by two-dimensional gel electro-
phoresis. (A) Control: addition of water instead of enzymes to the
reaction mixture. (B) Hb incubated with N-glycosidase F (cleavage
of N-linked sugars). (C) Release of O-linked sugars. (D) Release of
N- and O-linked sugars. New spots occurring due to the removal of
O-linked or O- and N-linked glycans are marked with white circles.
One spot, which is usually not found in Hb subunit patterns, is
labeled with an asterisk.
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3399
Molecular mass of Hb subunits
The reported molecular mass of Daphnia Hb didomain
subunits varies between 31 and 40 kDa [14,24]. Hb
proteins of twice this molecular mass have also been
reported for crustaceans (Lepidurus [36]; Daphnia pulex
[37,38]; Daphnia magna [15]), presumably resulting
from subunit dimerization. For Triops longicaudatus
and Cyzicus Hb subunits, molecular masses between
15 and 21 kDa have been found [32,39].
To determine the molecular mass of protein sub-
units, SDS ⁄ PAGE is often used because of its advanta-
geous properties (rapid and sample-saving). However,
a frequently neglected problem arises from the buffer
system used. A common protocol makes use of a Tris
buffer system [19]. Weber and Osborn [21] introduced

another buffer system (sodium phosphate buffer). For
Hb, the number of separated subunits is lower with
the Weber and Osborn method than with a Laemmli
SDS ⁄ PAGE protocol. For the subunits of Daphnia
pulex Hb, Dangott and Terwilliger [37] determined five
bands using the Laemmli protocol, but only two prom-
inent bands employing the Weber and Osborn proto-
col. In this study on D. magna Hb subunits, three
bands were found using the Laemmli protocol and
only a single band with the Weber and Osborn proto-
col (Fig. 4). Gielens et al. [40] have reported that
hemocyanin and some other proteins bind only 0.7 g
sodium dodecyl sulfate (SDS) per g protein in
Tris ⁄ HCl and Tris ⁄ glycine buffers, whereas 1.4 g SDS
per g protein is bound in phosphate buffers. Rochu
and Fine [20] concluded that Tris ions may be attrac-
ted by amino acid residues, which are negatively
charged at the pH values used in SDS ⁄ PAGE, and
accordingly, proteins may not be fully saturated with
the detergent, with the consequence that the lower
density of negative charges may not lead to a strictly
mass-dependent electrophoretic mobility. Moreover,
Tris ions may decrease the number of SDS monomers
in solution by promoting the formation of micelles.
Consequently, during Laemmli SDS ⁄ PAGE the separ-
ation of polypeptides is influenced not only by the
molecular mass but also by the degree of coverage of
surface charges, which depends on the amino acid
composition of proteins. Phosphate buffers, however,
do not specifically interfere with the binding of SDS

to polypeptide chains, permitting their saturation
with SDS [20]. Accordingly, a Weber and Osborn
SDS ⁄ PAGE protocol may better reflect molecular mas-
ses. The deviating electrophoretic mobility of Hb sub-
units in Tris-buffered gels despite similar molecular
masses may also be caused by differences in their pri-
mary structure. Actually, it has been reported for a
variant of serum prealbumin that a single-point muta-
tion, which results in methionine instead of threonine,
leads to a different migration behavior and an appar-
ently lower mass of the variant prealbumin form dur-
ing SDS ⁄ PAGE [41]. In addition, glycosylated proteins
are known to show unusual migration behavior during
gel electrophoresis [27]. The observed difference in
migration behavior of D. magna Hb subunits in buffer
systems according to Laemmli [19] compared to Weber
and Osborn gels [21] may therefore indicate a post-
translational modification of Hb subunits (see below).
To determine the molecular mass of D. magna Hb
subunits exactly, ESI-MS experiments were performed.
After deconvolution of the raw spectrum, a single peak
was high above background level. The molecular mass
of this major component was calculated to be
37.715 kDa, including two heme groups. The difference
between the detected mass and the molecular masses cal-
culated from amino acid sequences may be caused by
Fig. 8. GC mass analysis of carbohydrate
moieties of Daphnia magna hemoglobin
(Hb). Saccharides were released by
methanolysis and separated by GC. Compar-

ison with a mannose standard (gray-shaded
curve) identifies this saccharide as the dom-
inant glycan in D. magna Hb. A significant
increase of galactose and glucose in com-
parison to the background level [dashed
line ¼ maximum total ion current of the
blank (inset)] was also observed. One peak
marked with an asterisk could not be identi-
fied.
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3400 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
glycosylation (see below). Considering that the relation-
ship between Hb genes and Hb subunits is not yet com-
pletely determined, this post-translational modification
may also be the reason for the discrepancy between the
result of one dominating component in ESI-MS meas-
urements and the expected number of six gene products
(i.e. the maximum number assuming expression of all
known Hb genes [12]) or at least four different subunit
types that can be detected as main components by two-
dimensional electrophoresis in Hb from red animals
*
A
C
B
D
E
F
*
*

Fig. 9. Electron micrographs (A–D) and three-dimensional reconstruction (E–F) of Daphnia magna hemoglobin (Hb), which was purified by gel
filtration and negatively stained with 2% uranyl acetate for electron microscopy or was purified by chromatofocusing and negatively stained
with 5% ammonium molybdate containing 0.1% trehalose on holey carbon grids for three-dimensional reconstructions. (A) Hb of pale ani-
mals. (B) Magnification of a section in (A). (C) Hb of red animals. (D) Magnification of a section in (C). (Hb molecules showing the clover-leaf
structure are indicated by arrows. Ferritin molecules are indicated by asterisks. Bar, 50 nm.) (E) After a multireference alignment, matchable
images were treated by multivariate statistical analysis. Three examples of the resulting class averages are shown in (E) which correspond
approximately to the views of the three-dimensional reconstruction (F), showing the characteristic clover-leaf structure of red D. magna Hb:
presumed top (F, center) and two side views (F, left and right) of the three-dimensional reconstruction of D. magna Hb at sub-30 A
˚
resolu-
tion. [Note different scaling of molecules in (E) and (F).] In the center of the Hb molecule, a single mass was observed (F, center). (White
areas represent molecular masses.)
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3401
[42], respectively. The subunits contributing to the Hb
of red D. magna are mainly A, B, D and F, and accord-
ingly, more than one main subunit type would have been
expected in the ESI-MS spectrum. Although three of
these subunits (A, B and D), comprising almost 70% of
the total subunits present in Hb from red Daphnia [42],
seem to be of similar size in both gel electrophoresis sys-
tems used, their expected size differences should have
been resolved by MS. Again, glycosylation may contrib-
ute to the observed discrepancy. Different glycan struc-
tures seem to generate a complex mixture of masses
leading to a variety of charge states, which are difficult
to resolve by ESI-MS. Actually, under the experimental
conditions used for ESI-MS, only a single subunit type
could be detected reliably. It is most likely that this phe-
nomenon may also be affected by electrospray ioniza-

tion suppression, which is typically observed in ESI-MS
of extracts from biological samples [43]. However, the
value measured was used to calculate the number of sub-
units in the native aggregate.
Based on the molecular mass of macromolecule and
subunits, the number of subunits per macromolecule
can be calculated. For red D. magna, the molecular
mass of the native Hb complex (ultracentrifugation,
583 kDa; MALLS, 590.4 kDa, red, and 597.5 kDa,
pale) divided by the subunit mass (ESI-MS:
37.715 kDa) results in 16 subunits per Hb macromol-
ecule, independent of the method applied for the deter-
mination of the Hb complex mass. Because of an
underestimation of both macromolecule and subunit
mass (see above), Ilan et al. [14] came to the same con-
clusion of 16 subunits per D. magna Hb macromolecule.
Hb glycosylation
The mean molecular mass of D. magna Hb subunits
calculated from the nucleotide and the derived amino
acid sequences (Hb genes dmhb1–dmhb4 [11,12]) is
36.207 ± 0.027 kDa. Accordingly, the experimentally
determined value for the predominant peak found in
ESI-MS is 275 Da higher (red D. magna Hb subunits)
than the calculated value. Actually, the subunits of red
D. magna Hb were found to be glycosylated (Fig. 6)
using the Pro-Q Emerald 488 stain. Although Pro-Q
Emerald 300 dye is capable of detecting proteins with a
higher sensitivity (300–1 ng) and a broader dynamic
range (500–1000-fold), the Pro-Q Emerald 488 dye is the
most sensitive dye for detection of glycoproteins in gels,

when a laser-based gel scanner such as the FLA-2000 is
used for imaging [44]. For proteins with a high carbohy-
drate content (9–42%), the detection sensitivity is repor-
ted to be between 5 and 9 ng with a linear dynamic
range of 128–255-fold. Even glycoproteins with lower
carbohydrate content (3–7%) were successfully detected
(sensitivity 19 ng, linear dynamic range 64-fold) [44].
Protein isoforms that have the same amino acid
sequence, but different glycosylation profiles, often
appear as trains of spots on two-dimensional separa-
tions, which can differ in pI and⁄ or molecular mass
[45]. Hence, in a next step, enzymatic deglycosylation
was performed using different sets of N- and O-glyco-
sidases to remove only one type of glycan or com-
pletely remove all common glycans (Fig. 7). Additional
spots occurred when the Hb samples were treated with
enzymes releasing specifically O-linked glycans or with
all enzymes, respectively. After treatment with N-gly-
cosidase F, which cleaves only N-linked glycans, addi-
tional spots were not found. These results indicate that
the carbohydrates of Hb are O-linked.
This is in accordance with amino acid sequence
analyses for determination of the glycosylation type.
Whereas no N-linked glycosylation sites are present in
D. magna Hb [22], analyses using the NetOGlyc 3.1
server [23] revealed 11–12 potential O-linked glycosyla-
tion sites in Hb subunits exclusively in the pre-A seg-
ments. This server is reported to correctly predict 76%
of glycosylated residues and 93% of nonglycosylated
residues. It is intended for extracellular proteins and

can predict sites for completely new proteins without
losing its performance [23].
In order to identify the carbohydrates bound to Hb
subunits, saccharides were released by methanolysis
and analyzed by GC followed by MS (Fig. 8). Man-
nose was identified as the dominant sugar, whereas
galactose and glucose were found in smaller quantities.
Presently, directly O-linked mannose cannot be
removed enzymatically. However, the occurrence of
additional spots in two-dimensional gel electrophoresis
(Fig. 7) indicates (a) that some mannose is indirectly
bound to the protein, allowing a cleavage, and ⁄ or (b)
that the mobility shifts originate from the removal of
galactose and glucose. The fact that the spots A–D
and F were still present after 3 h of incubation with
deglycosylating enzymes can be explained by incom-
plete deglycosylation or the presence of mannose
directly bound to the proteins.
Although glycosylation is a common post-transla-
tional modification of the respiratory pigments of
invertebrates [46], Hb subunits of Daphnia pulex were
reported to be not glycosylated [38]. However, the ana-
lysis of distribution of glycosylation among taxonomic
groups showed that even closely related species may
not necessarily share close similarities in their glycan
diversity [47].
In conclusion, all experimental results concerning
the molecular mass of Hb subunits can be explained
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3402 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS

satisfactorily in the light of glycosylation. It can be
assumed that the molecular mass found by ESI-MS,
which was higher than the values calculated from the
amino acid sequences, is due to addition of a glycan
moiety. Although O-linked glycans are relatively small
compared to N-linked glycans, they can effectively
cover charges at the surface of a protein. Therefore,
this modification is suggested to influence the migra-
tion behavior of Hb subunits during gel electrophoresis
by interfering with binding of SDS, leading to three
bands or a single band during one-dimensional separa-
tions, depending on the buffer system used (Tris versus
phosphate buffer). As indicated by the alteration of
Hb subunit patterns after two-dimensional gel electro-
phoresis due to enzymatic deglycosylation, the pres-
ence of glycans influences isoelectric focusing of
subunits as well, supporting the idea that glycans affect
the net charge of Hb subunits.
Quaternary structure
Owing to its large size, the gross structure of a crusta-
cean Hb molecule can be assessed by electron micros-
copy. Ilan et al. [14] performed electron microscopy of
D. magna hemolymph proteins and detected a small
number of ring-like projections (diameter: approxi-
mately 14 nm), which they considered to be Hb. On
the basis of these electron micrographs and assuming
16 subunits per macromolecule, they discussed struc-
tural models for the multisubunit assembly, including
a cyclic and two dihedral structures containing either
both heterologous and isologous bonds or two types

of isologous bond. Based on pH dissociation experi-
ments, they favored a dihedral symmetry, D8, contain-
ing both heterologous and isologous bonds. This
model consisted of 16 subunits grouped in two layers
stacked in an eclipsed orientation, the eight subunits of
each layer occupying the vertices of a regular eight-
sided polygon. As this model was based on the small
number of ring-like projections, it may, however, not
be valid. Reporting difficulties in the electron micros-
copy of Daphnia Hb, the authors applied a contrast
enhancement technique (rotational photography) to
improve the resolution of objects with a rotational
symmetry. However, the major fraction of other mac-
romolecules visible in the preparation [14] was not
resolved by this technique. All Hb-utilizing animals
possess ferritin as iron-storage protein in the hemo-
lymph. In Nereis virens hemolymph, for example, a
small number of ring-like molecules (molecular mass
about 500 kDa; diameter approximately 10 nm diam-
eter) were also detected apart from a major fraction of
Hb [48]. This minor fraction was identified as ferritin
particles, due to their electron-dense iron cores.
Accordingly, the minor fraction of ring-like particles
within the hemolymph of D. magna (Fig. 9A,C,D)
may also represent ferritin molecules and not Hb.
Electron micrographs of the hemolymph of red
D. magna revealed predominant structures of tetramer-
ic symmetry (clover-leaf structures; see Fig. 9C,D,
arrows), which are supposed to represent red D. magna
Hb macromolecules. In pale D. magna hemolymph

(Fig. 9A,B), these specific structures were present only
to a much lower degree. As red D. magna Hb is com-
posed of a different set of subunits than pale D. magna
Hb [8,42], it is possible that both Hb isoforms also dis-
play a different quaternary structure.
After purification of red D. magna hemolymph by
chromatofocusing [removal of ring-like molecules
(‘ferritin’), but retention of high Hb quantities], numer-
ous different views of the Hb molecules were obtained
by negative staining of these molecules across holey
carbon grids, allowing a three-dimensional reconstruc-
tion by single-particle analysis. Negative staining tech-
niques have been widely used to obtain images of
macromolecules with high contrast. Although the stain
may invade aqueous channels, structural information
is basically limited to the shape of a macromolecule,
which may also be distorted due to air drying [49].
Despite these restrictions, this method is used in high-
resolution electron microscopy of macromolecules as
an important first step in identifying characteristic
views and has been used with great success in numer-
ous computer reconstructions of viruses and other
large macromolecular assemblies [49].
The gross structure of D. magna Hb turned out to
be much more complex than hitherto suggested
(Fig. 9E,F). Consisting of 16 subunits with a molecular
mass of 37.715 kDa, the macromolecule has the overall
shape of a square. Three-dimensional reconstructions
of subunits revealed by homology modeling [42] were
fitted into that of the macromolecule, but the resolu-

tion of the quaternary structure model made it difficult
to determine the exact positions of individual subunits
in the multimer. However, tight packing of subunits
was evident (data not shown).
Each side of the square is approximately 12 nm in
length, and a single mass in the center of the macro-
molecule was observed (Fig. 9F, center). This apparent
mass, however, may be an artefact, because in conven-
tional negative staining, the contrast medium may local-
ize preferentially on the surface of a molecule, leaving its
center more or less free of stain. In negative staining,
protein masses are visualized because of their lower elec-
tron density compared to the contrast medium. There-
fore, the incomplete filling of the molecule’s center with
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3403
ammonium molybdate ⁄ trehalose leads to a low electron
density, which thus may generate the impression of a
mass. In side views (Fig. 9F, left and right), the macro-
molecule appears as a compressed sphere with a height
of about 8 nm. However, flattening of the macromol-
ecule may have originated from negative staining and, in
reality, it may be more spherical.
A unique feature conserved in D. magna Hb chains is
the presence of an unusual N-terminal extension. This
extension is reminiscent of the 18-residue C-terminal
extension found in the two-domain Hb chain of a para-
sitic nematode, Ascaris suum , the so-called polar zipper
sequence. Initially, the extensions of individual subunits
were suggested to act as cement between the subunits in

the center of the octamer [50], and an equivalent func-
tion of the pre-A segments was discussed for D. magna
Hb subunits [11]. Detailed analysis of A. suum Hb
finally revealed that these extensions do not play a role
in stabilization of the quaternary structure once formed,
but rather function as intramolecular chaperones, aiding
assembly of the nascent Ascaris Hb octamer [51]. In fact,
the relevance of the pre-A segments in D. magna Hb
subunits seems to be determined by glycosylation.
Amino acid sequence analyses using the NetOGlyc 3.1
server revealed possible O-glycosylation sites only in the
pre-A segments. Since O-glycosylation is mainly a post-
translational and postfolding event, which affects the
structure of a protein by determining the secondary, ter-
tiary and quaternary structure (aggregation, multimeri-
zation) and occurs only at surface-exposed serine and
threonine residues [52,53], it can be assumed that the
pre-A segments are located on the surface of the mole-
cule. Concerning its biological impact, glycosylation is
suggested to be important for the folding, oligomeriza-
tion and transport of proteins and is reported to confer
protease and heat resistance to glycoproteins [53] as well
as to increase the solubility of proteins [52], as in the
case of vitellogenin from the decapod crustacean Cherax
quadricarinatus [54]. A localization of the pre-A seg-
ments in the center of the Hb macromolecule is also not
supported by the three-dimensional reconstruction, as
the apparent central mass is possibly an artefact.
Instead, the existing data indicate that these segments
may be involved in adjusting important biophysical

properties of the native molecule. For a definite elucida-
tion of the fine structure of D. magna Hb, however,
three-dimensional reconstructions based on macromole-
cules prepared by cryo-electron microscopy have to be
established.
The exceptional characteristic of D. magna of pursu-
ing a regulatory strategy to cope with changing oxygen
and temperature conditions by strong variations in the
quantity (concentration) and quality (subunit composi-
tion and oxygen affinity) of its respiratory protein
[55,56] has made D. magna an example of molecular
adaptation or acclimatization. The information avail-
able on D. magna Hb gene promoters and open read-
ing frames as well as protein subunits and subunit
composition [2,4,8,11,12,42] offers the chance to relate
the structure and function of this interesting molecule.
The present study has contributed to this approach:
(a) the determination of the molecular mass of the
native Hb complex and its subunits as well as the
number of subunits in the macromolecule; (b) evidence
for glycosylation of their pre-A segments, which may
support macromolecule assembly and provide addi-
tional functional qualities; and (c) a first reconstruction
of the native Hb complex.
Experimental procedures
Animals and Hb samples
Female D. magna Straus organisms were initially obtained
from the Staatliches Umweltamt, Mu
¨
nster, Nordrhein-

Westfalen, Germany and kept in laboratory culture for
many years. They were raised in iron-enriched (1 mgÆL
)1
ferrous iron [57]), standard culture medium [58] at 20 °C.
‘Pale’ D. magna, i.e. those with a low Hb concentration,
were raised under normoxic conditions (oxygen partial
pressure: 20.7 kPa) in a 40 L aquarium by gentle ventila-
tion with room air. ‘Red’ D. magna, i.e. those with an ele-
vated Hb concentration, were obtained by reducing the air
pressure inside closed 2 L glass vessels to 15% of atmo-
spheric pressure (oxygen partial pressure: 3.1 kPa). Animals
were fed daily with algae (Desmodesmus subspicatus). The
media were changed every 4 weeks.
Hemolymph was obtained by cutting off the animals’ sec-
ond antenna and collecting it with a 2 lL capillary tube
(minicaps; Hirschmann, Eberstadt, Germany). Determin-
ation of Hb concentration was carried out as described pre-
viously [5,6]. Gel filtration was performed on a Superdex
200 column (10 mm · 300 mm) equilibrated with 50 mm
Tris ⁄ HCl, pH 8.0 (for details, see [24]). Separation of Hb
isoforms by chromatofocusing was performed on a Mono-
Q HR10 ⁄ 10 column (Pharmacia, Uppsala, Sweden) using a
pH gradient between pH 8.1 and 4.2 (for details, see [42]).
Analytical ultracentrifugation
Sedimentation velocity and sedimentation equilibrium
experiments were carried out in a Beckman Optima XL-I
analytical ultracentrifuge (Palo Alto, CA) using an An-50Ti
rotor. Velocity runs were conducted at 20 °C at 116 480 g
using 0.2 mgÆmL
)1

Hb of red D. magna in 10 mm
ammonium acetate buffer (pH 6.7). Sample cells with 12 mm
double-sector charcoal-filled epon centerpieces and quartz
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3404 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
windows were used for all experiments. The absorbance of
the cells was scanned at 280 nm every 4 min during the run.
Velocity data were analyzed using the method of van Holde
and Weischet [59], as implemented in the program ultra-
scan 6.0. Sedimentation coefficients (s
20,w
) were corrected
for 20 °C and water. Sedimentation equilibrium runs were
conducted at 4 °C at 4660 g, 5900 g and 7280 g using protein
concentrations of 0.2 mgÆmL
)1
Hb of red D. magna in
ammonium acetate buffer. Sample cells with six-channel
charcoal-filled epon centerpieces and quartz windows were
used for all experiments. Samples were run for at least 24 h
at each speed. The absorbance of the cells was scanned at
280 nm, and it was assumed that equilibrium was achieved
when scans taken at intervals of 1 h showed no significant
change. Equilibrium data were analyzed with the program
ultrascan 6.0, employing the global fit routine to integrate
data taken at different speeds. For all calculations, a partial
specific volume of 0.749 cm
3
Æg
)1

was used, as published for
D. magna Hb by Ilan et al. [14].
MALLS
MALLS measurements were performed with a DAWN
EOS system (Wyatt Technology Corp., Santa Barbara, CA)
directly on-line with an HPLC system (Waters, Milford,
MA). Gel filtration was performed on a 1 · 30 cm Supe-
rose 6-C column (fractionation range from 5 to 5000 kDa;
Pharmacia) equilibrated with 0.1 m Tris ⁄ HCl buffer,
pH 7.0. The eluate (flow rate 0.5 mLÆmin
)1
) was simulta-
neously monitored with a photodiode array detector
(Waters 2996) and a refractive index detector (Waters
2414). The MALLS instrument was placed directly before
the refractometer to avoid back pressure on the instru-
ment’s cell. Chromatographic data were collected and proc-
essed with the astra software (Wyatt Technology Corp.).
The Zimm fit method was used for molecular mass determi-
nations [60]. In this method, the variation rate of the
refractive index as a function of concentration, dn ⁄ dc, was
set to 0.193 mLÆg
)1
(typical for human Hb [61]). BSA
monomer (Sigma, St Louis, MO) was used for normalizing
various detectors’ signals relative to the 90° detector signal.
Hb of red and pale D. magna (concentration 0.14–
0.93 mgÆmL
)1
; dissolved in 10 mm ammonium acetate buf-

fer, pH 6.7) purified by gel filtration or chromatofocusing
was used. The sample was kept at 4 °C until the elution,
which was performed at ambient temperature.
Analysis of subunit molecular mass by gel
electrophoresis
Two methods of molecular mass analysis by SDS⁄ PAGE
were employed. The multiphasic buffer system according to
Laemmli [19] was used with 3.6% stacking gels (0.125 m
Tris, 0.1% SDS, pH 6.8) and 12% separating gels (0.559 m
Tris, 0.1% SDS, pH 8.8). Electrophoresis buffer consisted
of 25 mm Tris, 250 mm glycine, and 0.1% SDS. The con-
tinuous buffer system according to Weber and Osborn [21]
was used with a 12% separating gel (0.1 m sodium phos-
phate, 0.1% SDS, pH 7.1) without a stacking gel. Electro-
phoresis buffer consisted of 0.1 m sodium phosphate and
0.1% SDS (pH 7.1).
Two micrograms of pale and red D. magna Hb dissolved
in sample buffer [0.5 m Tris ⁄ HCl, 3.5% SDS, 10% glycerin,
pH 6.8, containing bromophenol blue, and 0.1 m sodium
phosphate, 1% SDS, 1% dithiothreitol, 30% glycerin,
pH 7.1, containing bromophenol blue] were incubated at
95 °C for 10 min and loaded onto the gel. Minigels (about
85 · 60 · 1 mm) were run at a current of 15 mAÆgel
)1
for
70 min, 20 mAÆgel
)1
for 30 min and 30 mAÆgel
)1
for

150 min (Tris gel) and 105 min (sodium phosphate gel),
respectively. Gels were fixed in 20% trichloroacetic acid for
20 min and stained in a solution containing final concentra-
tions of 0.1% Coomassie R250, 30% methanol, and 10%
acetic acid for 30 min. Destaining was performed by diffu-
sion in a solution of 25% methanol and 10% acetic acid on
an orbital shaker, until protein bands were clearly visible.
ESI-MS
ESI-MS was performed under denaturing conditions to
determine the molecular masses of the subunits of D. magna
Hb. Electrospray data were acquired on an ESI-Q-TOF (Q-
TOF II; Micromass, Altrincham, UK) mass spectrometer
scanning over the m ⁄ z range 600–5000 at 1.5 sÆscan
)1
. Data
were accumulated over 3 min to produce the final spectrum.
Spectra were obtained on an Ouest-genopole
Ò
sequen-
cing ⁄ genotyping platform setup at Roscoff. Samples at a
concentration of 0.5 lgÆlL
)1
in acetonitrile ⁄ water (1 : 1,
v ⁄ v) containing 0.2% formic acid were introduced into the
electrospray source at 5 lLÆmin
)1
. The cone voltage (counter
electrode to skimmer voltage) was set to 60 V. Mass scale cal-
ibration employed the multiply charged series from horse
heart myoglobin (16951.5 Da; Sigma Cat. No. M-1882). The

raw ESI-MS spectra were deconvoluted using the maximum
entropy based software (maxent I) supplied with the instru-
ment [62], in order to find the approximate mass of each sub-
assembly (monomer and polymer). Hb fractions obtained by
chromatofocusing of crude extracts of red and pale D. magna
were desalted by washing with Milli-Q water and centrifuga-
tion in Amicon 3 kDa filter devices (Millipore, Bellerica,
MA) 10 times at 4 °C, before ESI-MS analysis. Molecular
masses are based on the atomic weights of the elements given
by IUPAC.
Analysis of Hb glycosylation and phosphorylation
Two-dimensional gel electrophoresis of D. magna Hb
Hb of red D. magna purified by gel filtration [24] was
diluted in rehydration solution containing 8 m urea, 2 m
T. Lamkemeyer et al. Structure of Daphnia magna hemoglobin
FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3405
thiourea, 2% CHAPS, 0.5% pharmalytes 3 ⁄ 10, and 65 mm
dithiothreitol to a final volume of 125 lL. Immobilized pH
gradient (IPG) strips (7 cm, pH 3–10, Biorad, Munich,
Germany) were actively rehydrated (50 V) for 12 h. After
focusing (maximum 4000 V, 22 000 Volthours), the IPG
strips were equilibrated for 15 min in 6 m urea, 2% (w ⁄ v)
SDS, 30% (w ⁄ v) glycerol, 50 mm Tris, pH 8.8, containing
1% dithiothreitol; this was followed by incubation in the
same solution, but with dithiothreitol replaced by 4% iodo-
acetamide, for an additional 15 min. The IPG strips were
then placed on 12% separating gels (thickness 1 mm) in the
second dimension (15 mAÆgel
)1
for 15 min, 30 mAÆgel

)1
for
70 min). The gels were silver stained or stained for glyco-
proteins, followed by staining with RuBPs (see below).
Glycoprotein staining
Hb of red D. magna (100 lg, Hb purified by gel filtra-
tion) was subjected to two-dimensional gel electrophor-
esis. For demonstration of glycosylated proteins, the
Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain kit
(Molecular Probes, Go
¨
ttingen, Germany) was used
according to the manufacturer’s protocol. Glycoprotein
staining was followed by total protein staining using the
fluorescence stain RuBPs, synthesized according to [63],
and staining was performed according to [64] or via sil-
ver staining. To prove the specificity of the staining tech-
nique, the CandyCane glycoprotein molecular mass
standard (Molecular Probes) ranging from 14 to 180 kDa
was used. When separated by electrophoresis, the Candy-
Cane standards appear as alternating bands correspond-
ing to glycosylated and nonglycosylated proteins.
Fluorescent signals were detected using an FLA-2000
laser scanner (Fuji Photo, Tokyo, Japan).
Enzymatic deglycosylation
For removal of carbohydrate moieties, the Glycoprotein
Deglycosylation Kit (Merck Biosciences, Schwalbach,
Germany), which contains all enzymes and reagents nee-
ded to remove all N-linked, all simple O-linked, and vir-
tually all complex O-linked oligosaccharides from

glycoproteins, was used according to the manufacturer’s
protocol. To analyze the type of glycosylation (N- or O-
linked sugars), 50 lg of Hb purified by gel filtration [24]
was incubated either with N-glycosidase F (removal of
N-linked sugars), or a mix of endo-a-N-acetylgalactosa-
minidase, a2–3,6,8,9-neuraminidase, b-1,4-galactosidase,
and b-N-acetylglucosaminidase (removal of O-linked
sugars), or a mixture of all five enzymes (removal of
N- and O-linked sugars), for 3 h at 37 °C. A sample in
which water was added instead of enzymes served as a
control. Deglycosylation was monitored by mobility shift
assays using separation of proteins by two-dimensional
gel electrophoresis.
GC ⁄ MS
For identification of carbohydrates, crude extracts of
D. magna were purified by gel filtration [24]. Eluates con-
taining Hb were desalted by washing with water and centri-
fuging using Nanosep spin columns (MWCO 10 kDa, Pall
Life Sciences, Ann Arbor, MI) and concentrated to dryness
in a rotational vacuum concentrator (RVC 2-25, Christ,
Osterode am Harz, Germany). An eluate of the gel filtra-
tion column eluting at the same time as the Hb was lyophi-
lized and analyzed by GC for the presence of saccharides
originating from the used buffer and ⁄ or from the column
material (blank). Carbohydrates were cleaved by methano-
lysis (0.6 m HCl in absolute methanol for 18 h at 70 °C),
and the methyl glycosides were trimethylsilylated [100 lL
of 1 : 1 BSTFA (bis(trimethylsilyl) trifluoroacetamide) ⁄ pyr-
idine for 1 h at 60 °C]. The derivatization solution was
directly analyzed by GC ⁄ MS (Agilent 6890 ⁄ 5973, Agilent,

Waldbronn, Germany) on a DB-5 (J+W, Folsom, CA)
capillary [25 m · 0.25 mm, film thickness (d
f
) ¼ 0.25 lm].
Individual saccharides were identified on the basis of their
mass spectra, their retention times and characteristic ratio
of the a- and b-anomers.
Analysis of phosphorylation
For the detection of possible phosphorylation, Hb of red
D. magna (purified by gel filtration [24]) was subjected to
one-dimensional gel electrophoresis. Staining of phosphor-
ylated proteins was performed using the Pro Q Diamond
Phosphoprotein Stain Kit (Molecular Probes) according to
the manufacturer’s protocol, followed by total protein
staining using the fluorescence stain RuBPs. In addition,
proteins were transferred onto PVDF membranes (Schlei-
cher & Schuell, Dassel, Germany) after one-dimensional gel
electrophoresis using a semidry western blot apparatus
(Peqlab, Erlangen, Germany) and incubated separately with
antibodies directed against phosphotyrosine (monoclonal
anti-phosphotyrosine, clone PY20, Sigma, Steinheim, Ger-
many) or phosphoserine residues (phosphoserine antibody
Q5, Qiagen, Hilden, Germany). Antibody binding was
detected using alkaline phosphatase. For MS, Hb of red
animals was digested with trypsin and phosphorylated pep-
tides were enriched using immobilized metal affinity chro-
matography with Fe
3+
,Ga
3+

and Zr
4+
as well as affinity
chromatography with TiO
2
. Nano-LC-MS ⁄ MS-measure-
ments were accomplished on a nano-HPLC (Ultimate sys-
tem, Dionex GmbH, Idstein, Germany) coupled to a linear
ion trap (QTrap 4000, Applied Biosystems, Framingham,
MA) operating in precursor ion scanning mode. The repor-
ter ion mass of 79 Da for the phosphate group in negative
mode was used for detection of Ser- ⁄ Thr-phosphorylated
peptides, and the reporter ion mass of 216.043 Da was used
for the immonium ion of phosphotyrosine in positive scan-
ning mode.
Structure of Daphnia magna hemoglobin T. Lamkemeyer et al.
3406 FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS
Electron microscopy and three-dimensional
reconstruction
Specimen preparation
Conventional negative staining with 2% uranyl acetate of
D. magna Hb purified by gel filtration [24] was performed
by the single-droplet procedure as described by Harris and
Horne [65]. For single-particle image processing, D. magna
Hb purified by chromatofocusing (for details, see [42]) was
prepared using the holey carbon negative staining method,
as described by Harris and Scheffler [66]. The Hb concen-
tration was optimized by direct electron microscopic assess-
ment of the distribution of particles embedded within
the thin film of negative stain (5% ammonium molyb-

date + 0.1% trehalose) across the holes of the grid.
Electron microscopy
A Tecnai 12 transmission electron microscope (with LaB
6
filament) and a Zeiss EM900 were used. Electron micro-
graphs were recorded on Kodak 4489 electron image film.
The instrumental magnification was 49 000-fold.
Digitization and image processing
Electron micrographs were digitized using a Primescan
drum scanner (Heidelberger Druckmaschinen, Heidelberg,
Germany) at a resolution of 2700 d.p.i. The resulting reso-
lution ratio was 2.855 A
˚
Æpixel
)1
in the digitized micrograph.
Image processing was performed within the imagic 5 soft-
ware package (Image Science GmbH, Berlin, Germany).
Single-particle analysis was started with about 7500 single
particles from 10 different electron micrographs. Because of
an unequal background, a bandpass filter was used to buf-
fer noise and remove brightness shifts from the single-parti-
cle images. For equalization of transverse and rotational
distortions, a multireference alignment (MRA) was per-
formed [67]. The aligned and matchable images could be
treated by multivariate statistical analysis (MSA) [67,68].
The resulting class averages, each based on about 12 single
particles, showed randomly orientated molecules with much
better quality than the original images. For three-dimen-
sional reconstruction, a Euler search was performed using

different symmetry features, to calculate the relative orien-
tation (Euler angles) of the class images. From determined
relative spatial angles, the three-dimensional reconstruction
was conducted. The process was repeatedly performed by
iterative improvement of the reconstruction by quality esti-
mation during every single step of the process.
Acknowledgements
The technical assistance of Marita Koch, Ina Buchen
and Nicole Sessler is gratefully acknowledged. We
thank Professor Maier, University of Tu
¨
bingen, for the
synthesis of ruthenium II tris(bathophenanthroline di-
sulfonate). The excellent help of Dr Gnau and
Dr Buckenmaier, Proteom Centrum Tu
¨
bingen (PCT),
concerning molecular weight determinations by MS is
gratefully acknowledged. We also thank Dr Sickmann
and Jo
¨
rg Reinders from the Protein Mass Spectrome-
try and Functional Proteomics Group (Rudolf Vir-
chow Centre, University of Wu
¨
rzburg) for their
expertise in analysis of phosphorylation by MS.
Franck Zal and Morgane Rousselot would like to
thank the Conseil Re
´

gional de Bretagne and CNRS
for their financial supports. The Proteom Centrum
Tu
¨
bingen is supported by the Ministerium fu
¨
r Wis-
senschaft und Kunst, Landesregierung Baden-Wu
¨
rt-
temberg.
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